Serum is the liquid portion of whole blood which remains after blood is allowed to clot. Devoid from the serum are the whole blood components which are consumed or entrapped during the clotting process namely red blood cells, white blood cells, platelets and the blood coagulation factors. Thus, serum includes all of the proteins not used during clotting and also includes sugars, fats, enzymes, antibodies, antigens, hormones, charged particles (i.e., electrolytes) and exogenous substances (e.g., drugs and microorganisms). Serum is, therefore, the preferential test substrate on which to perform clinical tests used to diagnose and monitor muscle and organ function, metabolic balances, and basic physiologic functions. Also serum is preferred to perform other analytical tests such as enzyme, electrolyte, protein, and glucose assays since the interference of unwanted substances has been removed through the clotting process.
Serum is obtained by centrifugation of clotted blood. In the past, the production of serum from whole blood has been a passive process in which freshly collected blood is added to a glass test tube and allowed to clot. Alternatively, other serum tubes may contain silica or ellagic acid to stimulate the coagulation cascade. Blood, once removed from the body, has a natural tendency to clot and its exposure to a surface such as glass promotes clotting in a more efficient manner. Contact with a glass surface causes the activation of coagulation factors which interact in a mechanism commonly referred to as the coagulation cascade. In this process, an inactive coagulation factor is chemically converted to an active enzyme which subsequently converts yet another inactive precursor. The end result of the coagulation cascade is a conversion of the soluble plasma protein fibrinogen, to an insoluble protein, fibrin, whereby the fibrin clot entraps the white cells, red cells, and platelets forming a solid gelatinous mass. Substances not consumed in the process, such as those described above, remain free of the gelatinous mass and are found in the liquid matrix, i.e. the serum.
The passive clotting process described above causes several problems. While blood from normal healthy individuals may clot in 30 minutes or longer in a glass test tube, blood from sick individuals who may have deficiencies of coagulation proteins or from patients who are receiving anticoagulation therapy (i.e., oral anticoagulants or heparin) may require extensive time to clot (i.e., 2-8 hours). Consequently, there has been a delay associated with the obtaining of blood specimens and the performance of the analytical tests, thereby affecting the ability of the clinician to quickly provide optimal patient care. In addition, the blood from individuals with deficiencies of coagulation proteins or patients receiving anticoagulation therapy may never form a complete and adequate fibrin mass. For example, incomplete clotting in heparinized blood specimens results in a poor quality serum substrate upon which to perform the chemical test. Furthermore, serum from heparin anticoagulated blood, which may not have clotted initially, may begin to clot once placed in the analytical device, thereby clogging the system and causing an instrument shutdown.
In order to improve the predictability and uniformity of the clot forming process, technicians have routinely added the clot promoting agent thrombin and/or a closely related enzyme with “thrombin-like” activity (e.g. batroxobin) to the blood specimen. Thrombin actively converts fibrinogen to fibrin, thereby forming the clot more efficiently then the slower glass-activated clotting process. One drawback to using thrombin and/or another enzyme in a blood collection tube is that these proteins are not as stable as the silica-based contact pathway activators. For example, exposure to moisture and elevated temperatures can inactivate thrombin. However, silica-based methods have often failed to give complete blood clotting resulting in a partially clotted material that could interfere with the testing of blood samples such as serum obtained from whole blood.